The human adrenal cortex acts as a compound endocrine gland secreting mineralocorticoids and glucocorticoids. These steroids arise from distinct zones of the adrenal cortex that have both morphologic and biochemical differences. The long-range objectives of the proposed research are to define the mechanisms causing the functional zonation of the adrenal, which be traced to the zone-specific expression of the enzymes involved in adrenal steroidogenesis. This is particularly true for CYP11B2 (aldosterone synthase) and CYP 11B 1 (steroid 11 beta-hydroxylase) that are responsible for the final steps in the biosynthesis of aldosterone and cortisol and exhibit zone-specific expression in the glomerulosa and fasciculata, respectively. The mechanisms causing differential expression of CYP11B1 and CYP11B2 have not been defined due in part to the lack of appropriate in vitro model systems. Recent development of a human adrenocortical cell line (NCI-H295R) that grows as a monolayer and continues to produce mineralocorticoids and glucocorticoids has overcome this problem. The availability of these cells has allowed the applicant to propose three specific aims directed at defining the molecular mechanisms that control the zonal expression of human CYP 11B 1 and CYP 11B2. The goals of Specific Aims 1 and 2 are to identify specific cis-regulatory elements and trans-acting factors that enhance expression of human CYP 11 B2 in the glomerulosa and repress expression in the fasciculata. This will be accomplished by defining enhancer and repressor elements in the CYP 11 B2 5-flanking DNA and intronic sequences followed by identification of the trans-acting factors that bind these elements. The goals of Specific Aims 3 are to define the cis-elements and trans-acting factors that activate or inhibit CYP 11B 1 gene transcription and cause its zone-specific expression within the adrenal cortex. These newly defined genes will be studied with regard to their involvement in the expression ofCYP11B1 and CYP11B2 as well as their regulation in adrenal cells. The completion of these aims would add considerably to the understanding of adrenal zonation as well as the molecular mechanisms regulating two key enzymes involved in the synthesis of mineralocorticoids and glucocorticoids.